The proteome can be defined as the entire complement of proteins, including the post-translation-modified proteins, produced within an organism, tissue type, or a cell. Proteomics is a study of such entire protein complements, particularly their qualitative and quantitative changes in structure and expression resulting from the stimuli, stress and function of a living organism.
In order to address such structural and expressional changes in a partial or entire complement of proteins within a cell, numerous methods for high-multiplex biomarker quantification have emerged from several major categories of technologies: 2-dimensional electrophoresis, mass-spectrometry and immunoassays. Those technologies have already enabled the gathering of an unprecedented level of functional proteomic information as they have answered a large number of critical questions regarding multiple types of human diseases and environmental impact on human health. However, these methods face major challenges in meeting the clinical requirement for accuracy, reproducibility, sensitivity and specificity with respect to high throughput multiplex detection. Particularly, it remains very difficult for many of these methods to simultaneously detect and accurately quantify multiple samples of high-multiplex protein biomarkers that are run in parallel, simultaneously.
Many Double-Antibody-Sandwich Enzyme-Linked Immunosorbent Assays (DAS-ELISA) for a single biomarker have already been approved by the F.D.A. for clinic diagnostic measurement of multiple samples, due to the fact that these assays can pass the vigorous trials and tests for reliability, accuracy, sensitivity and specificity at multiple clinic and research centers. In the past decade, a huge effort has already been spent to incorporate the principles of the single-biomarker ELSIA into multiplex biomarker immunoassays for the testing of multiple samples using the analogue of microarray based genomic technology. Three major categories of ELISA microarrays currently exist: (1) planar slide arrays, (2) sphere bead arrays and, (3) micro-fluidic chip arrays. The multiplex detection with respect to micro-fluidic chips is in its infant stage and has very little data published as of yet.
In general, ELISA microarrays utilize some of the principles and antibody materials of traditional DAS-ELISA. For example, capture-antibodies are typically selected for having a first region that will couple to the surface of a solid substrate (e.g., planar slide or bead) and another region capable of binding to one of the targeted biomarkers that is potentially present in the sample. After first binding to the substrate, the capture-antibody is exposed to the sample and the biomarker(s) present therein. After allowing the biomarkers present in the sample to bind to the targeted regions of their respective capture-antibodies, a fluorescent or chemiluminescent-linked detect-antibody can be added to the substrate and allowed to bind to the exposed region (unbound region) of the biomarker to form a sandwich-like complex on the surface of the microarray support substrates. The intensity of the fluorescence or chemiluminescence signal from the complex can then be quantified, in order to estimate the concentration of the target biomarkers in the sample using an imaging device. This can be done by comparing the sample's signal to a known, or standard, signal.
Importantly however, previous multiplex-microarrays significantly deviate from the essential, original procedure of traditional single-biomarker DAS-ELISA. For example, the image-based signal quantification method is different from spectrometry used by traditional DAS-ELISA. Particularly, because the fluorescent or chemoluminescent signals on the surface of microarray substrate matrix are non-homogeneous, non-uniform and non-even, quantification by an imaging device is much less reproducible and sensitive than the counterpart from a homogeneous enzyme-substrate solution by spectrometer. Additionally, with respect to open microarrays (slides and beads), because multiple types of multiplex detect-antibodies are pre-mixed before they are allowed to probe the captured biomarker complexes, one of the detect antibodies may potentially form a non-specific sandwich complex with an unintended captured biomarker, thus indicating a false positive. This problem does not occur with traditional DAS-ELISA because the single captured-biomarker assay is only probed with one type of detect antibody. Furthermore, in planar slide microarrays, as each sample is processed on a microarray slide, each of the samples and their standard controls have to be processed separately on their own individual microarray slide. In practice, it is very difficult to simultaneously process the multiple samples and standard controls in parallel during the incubation and washing, probing and quantifying procedures, especially if the sample volume is in short supply. In beads based microarrays, the same biomarker is not simultaneously quantified among all of the samples and their standard controls in parallel. Thus, with respect to bead based arrays, the higher the number of multiplex biomarkers there are to be quantified, the wider the time gap will be for the quantification of the same biomarker.
Clearly, planar slides-based and sphere beads-based ELISA microarray technologies confine their users to carefully balance between their needs of biomarker multiplexing capacity (significance of proteomic bias), high throughput capacity (total number of standards and samples) and quantification reliability (related to accuracy, reproducibility, specificity and sensitivity). Other problems of those microarrays often happen after the printing of slide microarrays or the coupling of sphere beads with the capture-antibodies to the support surface. These problems can include the inactivation of antibodies due to storage instability, the changing of binding affinity between biomarker, capture antibodies, detect antibodies and support substrate surface due to inconsistent timing and handling.
To resolve the above-mentioned challenges, one of the general objectives herein is to maintain, or substantially maintain, the accuracy, reproducibility, specificity and sensitivity of biomarker quantification of traditional DAS-ELISAs during multiplex biomarker quantification of multiple small volume samples. The teachings herein encompass both new materials and methods related to a new multiplex microarray.